HUT63658A - Process for reducing the enantioselectivity of candida lipase and for producing immobilized candida lipase - Google Patents

Description

The present invention relates to a process for the preparation of immobilized lipase from a microorganism of the Candida genus and to its use for enethioselective esterification.

Immobilized lipase is prepared by reacting a solution of a lipase with an epoxide-activated macroporous support, and the lipase crosses the epilone amino group of the lysine contained therein.

By N-alkylation it is covalently bonded to the support through the opened epoxide bonds. The immobilized lipase undergoes enantioselective transesterification in the presence of an enol moiety with an enantiomeric mixture of alcohol containing at least one asymmetric center in its molecule.

Immobilization also increases the stability of the enzyme during repeated use.

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Budapest International Patent Office H-1061 BudapestL Dalszínház u. 10. In the winter: 153-3733, Fax: 153-3664

63 658

DISCLOSURE

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A method for increasing the enantioselectivity of Candida lipase and for preparing immobilized Candida lipase

Chemie Linz GmbH, LINZ, AUSTRIA Inventors: Dr. Furt Kurt, GRAZ,

BERGER Brigitte, GREIFENBURG,

AUSTRIA

Date of filing: 28.08.1992

Priority: 30.08.1991 (A 1715/91) AUSTRIA

The present invention relates to a process for increasing the enantioselectivity, activity and stability of a lipase from a microorganism of the Candida genus by an enzymatic transesterification process comprising the step of enetloselectively esterifying a chiral alcohol containing at least one asymmetric center in the molecule with an enol ester.

Chiral, enanthomerically pure alcohols are of great importance for various applications, such as the synthesis of pharmaceuticals or agrochemicals. They may be prepared, for example, by enethioselective transesterification, in which the enantiomeric mixture of a chiral alcohol is reacted with a hydrolase in the presence of a carboxylic acid ester. Because the hydrolases generally catalyze both the upstream and downstream reactions, the desired final product under these conditions will usually only be produced very slowly and with insufficient optical purity. M. Degueil-Castaing et al., Tetrahedron Letters 28 (9). 953-954 (1987) therefore recommend the use of an enol ester as the carboxylic acid ester, in which case the reaction cannot take place. Zakrzewska et al. (Acta Med.Pol., 2.9 (1-2), 44 (1988)) disclose that aldehydes formed from the enol ester space during transesterification deactivate the enzyme by reacting end-functional groups of the enzyme. EP-A-0 321 918 describes a process for the separation of enzymatic racemates of an enantiomeric mixture of a racemic alcohol with a vinyl ester or a vinyl ester in which the liberated aldehyde cannot deactivate the lipase. However, it has been found that the repeated use of a Candida lipase in such a process results in a very rapid loss of both activity and selectivity.

C.J.Grey et al., Enzyme Microbiol. Technol., 12, 800-807 (1990)] that the stability of a Candida cyundracea lipase can be increased by various immobilization techniques for repeated use. However, transesterification using enol esters is not mentioned.

Unexpectedly, it has been observed that both the stability, activity and, unusually, selectivity of a Candida lipase are enhanced by the use of an epoxide-activated macroperoxide-bearing macroscopic amine group of lysine in the lipase prior to use of the lipase in a transesterification process using enolesters. by immobilization by reaction with epoxide groups on N-alkylation. With this immobilization, both the lipase resistance to aldehydes and its activity and substrate selectivity increases with non-immobilized lipase, and the substrate selectivity is not reduced upon repeated application but remains completely constant.

The invention thus relates to the production of an immobilized lipase from a microorganism of the genus Candida, characterized in that the epilone amino groups of the lysine in the lipase are bound by N-alkylation via the opened epoxide groups of an epoxide-activated macroporous support, thereby immobilizing the lipase.

Lipase from a Candida genus includes both crude suspensions and purified enzyme fractions of the Candida genus. Species of the genus Candida (C.) include C.antarctica, C.rugósa, C.cylindracea. Preferably, lipase from C.cylindracea is used. Lipases from the Candida genus are commercially available in varying degrees of purity.

An epoxide-activated macroporous carrier is used to immobilize the lipase. It is prepared, for example, by the slurry polymerization of vinyl acetate and a vinyl acetate copolymerizable monomer, such as N, N'-divinyl ethyleneurea, in water.

by partial saponification of the acetate groups to the hydroxy groups and subsequent reaction with epichlorohydrin, which reacts with the free hydroxy groups to form an epoxide ring. This results in the formation of spacer groups of reactive epoxide groups in the polymerization, which are suitable for coupling the support to various substrates. Such carriers and their preparation are described, for example, by K. Burg et al., Angew. Makromol. Chem., 157, 105-121 (1988)]. These are commercially available. Particularly preferred carriers are Eupergit C (Rohm Pharma, Germany) or VA-Epoxy-Biosynth (Riedel de Haen, Germany).

ANGlazer (The Proteins, H. Neurath and RLHill, Eds., Academic Press, London, 1976, Vol. 2, pp. 1-109) discloses that, when such enzymes are immobilized, the epilone amino groups of lysine react primarily with the epoxide groups of the support. , during which the amino groups of lysine are alkylated.

Thus, the lipase of the present invention is alkylated on the epoxylamino groups of the lysine of the lipase by the epoxide groups of the carrier, i.e., it is covalently bound to the carrier.

The present invention therefore relates to a process for the preparation of the immobilized lipase discussed above. In the preparation, an epoxide-activated macroporous support is contacted with an aqueous solution of a Candida lipase enzyme at a temperature ranging from 15 ° C to a lipase deactivation temperature, preferably 18 ° C to 30 ° C, for example in a shaking flask. The ratio of carrier to lipase should be chosen such that the free amino groups of the lysine in the reaction mixture per group are · · ·

- 5 epoxy groups of the support. Preferably, about 0.05 to 0.1 g of lipase is used per gram of carrier. The lipase may be in pure water, dissolved in a buffer or saline. The epilone amino groups of the lysine in the lipase react with the epoxide groups on the support during N-alkylation to form a covalent bond between the lipase and the support. After completion of the reaction, the immobilized lipase, optionally after addition of salts, such as sodium chloride, to the reaction mixture is filtered and washed with buffer. The immobilized lipase may be stored in the buffer either wet or dried until use.

The lipase thus prepared can be used for the enantioselective esterification of enantiomeric mixtures of chiral alcohols using enol esters.

The invention therefore relates to a process for enantioselective esterification of an alcohol containing at least one asymmetric center in a molecule, characterized in that the alcohol is esterified in the presence of an enol ester and a lipase from a microorganism of the Candida genus, a lipase on an epoxylamino group of lysine in lipase. -activated macroporous support is covalently bonded by N-alkylation with the opened epoxide bonds and the resulting ester of the chiral alcohol and optionally the unreacted alcohol is separated from the reaction mixture.

The alcohol to be reacted has at least one asymmetric tracetrum and is therefore optically active. • a

- 6 in the form of mixtures of enantiomers or mixtures in which one or the other enantiomer is enriched.

The immobilized lipase of the present invention reacts with both primary and secondary alcohols. The alcohol may also be a divalent alcohol having two asymmetric centers in a molecule, in which case mixtures of R, S, S, R, R, R or S, S alcohols may occur. It is preferred to use as secondary substrates secondary alcohols containing an asymmetric center in the molecule.

As enol esters, for example, the enol esters disclosed in EP-A-0 321 918 can be used. Preferably vinyl esters of lower organic acids are used, in particular vinyl acetate, vinyl propionate, vinyl butyrate.

The reaction is carried out using from 2 to 30 g, preferably 6 to 10 g, of immobilized lipase and at least 5 equivalents of enol ester per gram of chiral alcohol-containing enantiomeric mixture, optionally mixed with an inert diluent under reaction conditions and stirring or shaking at 15 ° C. to a lipase deactivation temperature, preferably at room temperature.

The reaction can be carried out in a diluent under the reaction conditions, or the enol ester used for the transesterification itself can be used as a diluent and in large excess. Preferably, the reaction is carried out in an enol ester used in the reaction, rather than in a diluent employed under the reaction conditions.

• * · • · · · · · · · · · · · · · · ·

The reaction is conveniently carried out at a temperature where it shows the highest activity of the lipase. This temperature is usually specified by the manufacturer or supplier or simply determined experimentally. Depending on the alcohol mixture used, the enol ester and the specificity of the lipase used, the reaction predominantly yields R or the predominantly S-, or predominantly R, S, or predominantly S, R ester of the alcohol used, and the corresponding S- or the R or S, R, R, R, S- _Z R, R or S, S-alcohol are not or only slightly reactive.

The progress of the reaction is monitored by appropriate and known methods in enzyme chemistry, such as gas chromatography.

The reaction is terminated when the desired enantiomeric purity of the product is achieved and the reaction mixture is worked up. The immobilized lipase is filtered off and the mother liquor is evaporated at a suitable operating temperature, during which the desired products can be isolated. The desired product can be separated from the reaction mixture by extraction, distillation and chromatography in the usual manner.

In a preferred embodiment, the racemic mixture of an alcohol is mixed or shaken with Candida cylindracea lipase immobilized by VA-Epoxy-Biosynth (Riedel-de-Haen) in vinyl acetate. After the desired excess of enantiomeric R or S or R, S or S, R ester is formed, the enzyme is filtered off and the desired products are separated by distillation or chromatography.

• " the • · · · · · • · · · · • · · · · · ·

Our experiments have shown that the lipase resistance to acetaldehyde is increased in both activity and selectivity compared to a non-immobilized lipase. Particularly surprising was the more than 5-fold increase in selectivity, which remained completely constant upon repeated use of the immobilized lipase.

The immobilized lipase and its use in the enantioselective esterification of the present invention is a state of the art.

Example 1:

VA-Epoxy-Biosynth (Riedel-de-Haen, Germany) (10.0 g) was suspended in 70 ml of 0.1 N phosphate buffer, pH 7.0, and 300 mg of Candida cylindracea lipase (AY-30, Amano Pharm. Co. ., Japan) and shake at 27 ° C and 80 rpm for 3 days. Sodium chloride solution (70 mL) was added, followed by filtration, and washed twice with 30 mL each of 0.05 N phosphate buffer (pH 7.0) and dried.

The specific activity of the immobilized lipase thus obtained was determined by titration of the acetic acid obtained with hydrolysis of triacetin in 0.1 N phosphate buffer, pH 7.0, with 1 M sodium hydroxide solution, and 5.70 / 2 mol * min. g. The specific activity of the non-immobilized lipase was 13-1 mol * min * g between identical volumes.

Example 2:

g (9 mmol) of racemic endo-norborn-5-en-2-ol 200 mg of Candida <RTI ID = 0.0> (AY-30, </RTI> Amano) Pharm. Co., Japan) and shaken with 10 ml of vinyl acetate at 20 ° C and 200 rpm. The progress of the reaction is monitored by gas chromatography. The reaction was quenched after 4 hours. The lipase was filtered off and the mother liquor was concentrated in vacuo. Purification of the residue by conventional column chromatography on silica gel gave (+) - endo-norborn-5-en-5-yl acetate and (-) - endo-norborn-5-en-2-ol.

Measurement results for lipase activity, conversion and enantiomeric ratio are summarized in Table 1.

EXAMPLE 3;

The reaction is carried out as described in Example 2 except that the immobilized lipase prepared in Example 1 is used.

Measurement results for lipase activity, conversion and enantiomeric ratio are summarized in Table 1.

- 10 - «4 · • · · • 9 • · • · · · * «44« * ···  · · · · • · · · • · · · · · Table 1 THE Llpáz Nude. % EE % EE s (-) Alk. (+) Est. First Sum. 39 51.4 66.5 8 Second Sum. 1 11.0 54.0 4 Third Sum. - - - - First Imm. 100 62.2 91.3 42 Second Imm. 50 66.0 91.7 42 Third Imm. 23 59.1 91.8 42 THE table abbreviations used meaning a following: THE: applications number Sum. : comparison I: Immobilized lipase Nude. relative activity, defined by reaction down running a reaction first 2 0% —a. slope compared to

(tangent method).

% ee (-) Alk: enantiomeric excess of unreacted (-) - endo-norborn-5-en-2-ol% ee (+) Est: enantiomeric excess of (+) - endo-norborn-5-en-2-ol formed

The respective enantiomeric excess was determined by gas chromatographic separation of the corresponding menthyl chloroformate prepared by derivatization with (-) - menthyl chloroformate according to B. Berger et al., (1990) Tetrahedron: Asymmetry, 1, 541-546. .

S: Enantioselectivity of the reaction as determined by Chen et al., 1982, J. Am. Chem. Soc. 104, 7294-7299.

Claims (10)

CLAIMS 1. A lipase from a microorganism of the genus Candida, characterized in that the epilone amino groups of the lysine in the lipase are covalently bound by N-alkylation through the opened epoxide groups of an epoxide-activated macroporous support, whereby the lipase is immobilized. 2. The lipase of claim 1, wherein the macroporous support is prepared by slurry polymerization of vinyl acetate and a monomer in water which can be copolymerized with vinyl acetate, followed by partial saponification of the acetate into hydroxy groups and subsequent reaction with epichlorohydrin. reacts with the free hydroxy groups to form an epoxide ring. 3. A method for immobilizing lipase from a microorganism of the Candida genus comprising reacting an epoxide-activated macroporous support with a solution of Candida lipase in water, buffer or saline at a temperature ranging from 15 ° C to a lipase deactivation temperature, when covalent bonds are formed between the epilone amino groups of the lysine in the lipase and the opened epoxide bonds of the epoxide-activated macroporous support by N-alkylation. 4. The process of claim 3, wherein the macroporous support is reacted with 0.05 to 0.1 g of lipase per gram. Use of a lipase according to claim 1 for the enantioselective esterification of a chiral alcohol using an enol ester. • 4 · 4 «φ *** ·· * · · * ·· • · '·· *« · • 4 4 * * · •• «4 · ·« »· <4 6. A process for the enantioselective esterification of a chiral alcohol which comprises esterifying the alcohol in the presence of an enol ester and a lipase from a microorganism of the genus Candida which is epoxide-activated by macroporous epoxide activated by epoxide-activated macroporous support through the epilone amino groups of lysine. and the resulting ester of the chiral alcohol and optionally the unreacted alcohol is isolated from the reaction mixture. 7. The process of claim 6 wherein at least 5 equivalents of enol ester per gram of chiral alcohol and 2 to 30 grams of immobilized lipase are reacted. The process according to any one of claims 6 or 7, wherein the diluent for esterification is the enol ester which is also used as the reaction partner. 9. A process according to any one of claims 6 to 8, wherein the enol ester is vinyl acetate, vinyl propionate or vinyl butyrate. 10. The method of any one of claims 6 to 9, wherein the lipase is a lipase from a microorganism of the species Candida cylindracea.